This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. While most cancer immunotherapies have focused on eliciting specific CD8+ cytotoxic T lymphocyte killing of tumor cells, a mounting body of evidence suggests that stimulation of anti-tumor CD4+ T cell may be required for highly effective therapy. Several MHC class II-restricted tumor antigens that specifically activate such CD4+ helper T lymphocytes have now been identified, including one from a melanoma tumor that is caused by a single base-pair mutation in the glycolytic enzyme triosephosphate isomerase (TPI). This mutation results in the conversion of a threonine residue to isoleucine within the antigenic epitope, concomitant with a greater than five log-fold increase in stimulation of a CD4+ tumor-infiltrating lymphocyte line (TIL 1558). Different TCRs from the oligoclonal TIL 1558 line, including E8 and G4, have shown the ability to preferentially recognize the mutant TPI peptide presented by HLA-DR1. In order to understand the structural basis for enhanced CD4+ T cell recognition of the mutant peptide, we have obtained the crystals of TCR E8 bound to both wild-type and mutant TPI antigens presented by HLA-DR1. These E8/TPI/HLA-DR1 complex structures represent the first structural analysis of tumor antigens capable of stimulating CD4+ T cells.